Blocking TLR-Activation of Regulatory T cells Slows Disease in ALS Amyotrophic lateral sclerosis (ALS) is a devastating, rapidly progressive neurodegenerative disease. Our recent data demonstrate that regulatory T cells (Tregs) slow disease progression rates in both ALS patients and the mutant (m) SOD1 mouse model of ALS. In ALS patients, numbers of Tregs in blood of rapidly progressing patients are reduced compared to slowly progressing patients; both numbers of Tregs and mRNA expressions of CD25, FoxP3, IL4 and TGF inversely correlate with rate of progression. In fact, low FoxP3 mRNA levels not only predict future rapid progression rates of ALS patients, but also predict reduced survival. In the mSOD1 mouse model, numbers of Tregs are increased during the slow phase, during which time microglia display an alternatively activated (M2) phenotype. However, eventually neuroprotection is lost; numbers of Tregs decline and the microglia switch to a classically activated (M1) phenotype. Preliminary results indicate that this switch may at least partly be due to an accumulation of toxic misfolded rogue proteins and their activation of toll-like receptors (TLRs). Misfolded toxic proteins, such as SOD1 and TDP-43, added to microglia/motoneuron co-cultures activated the TLR/CD14 signaling pathway, resulting in microglial activation, NF?B and p38 activation, escalating release of pro-inflammatory factors (including NOX2), and increased neurotoxicity. These toxic rogue proteins also activate TLRs on T cells inhibiting their suppressive functions. We hypothesize that blocking TLR activation of Tregs could substantially slow disease in ALS. Our preliminary results demonstrate that blocking TLR-activation of Tregs dramatically slows disease in mSOD1 mice. CD4+CD25High Tregs isolated from mSOD1/TLR2-/- mice dramatically slow disease when transferred into our mSOD1/RAG2-/- mice, extending disease duration an unprecedented 122% or 2.24 fold compared to mSOD1/RAG2-/- mice. In addition, we have demonstrated that human Tregs not only survive in the mSOD1/RAG2-/- mouse - so can be used to evaluate treated human Tregs - but are as effective as, or more than mouse Tregs at slowing disease when transferred into our mSOD1/RAG2-/- mice. Our goal is to test our novel hypothesis using the following aims: 1) To identify the siRNA most effective at reducing TLR2 activation of human Tregs by a) isolating Tregs from ALS patients, transfecting the Tregs with siRNAs and subsequently evaluating TLR2 and FoxP3 expressions, and b) verifying in vitro that TLR2 activation is reduced after addition of TLR2 ligands and that transfected Tregs retain their suppressive functions examining Teff expansion. 2) To verify these siRNA-transfected Tregs retain their suppressive functions in vivo using our mSOD1/RAG2-/- mice by a) transferring the TLR2-inhibited Tregs to our mSOD1/RAG2-/- mice and evaluating disease progression, b) examining the spinal cord, lymph nodes, and blood in these mice throughout disease for neural injury, inflammation, and T cells. These experiments will lead to the identification of the most effective human TLR2 siRNAs and treatment protocols which can be directly translated to ALS patients.